Resumo

The highly infective entomopathogenic fungus Metarhizium anisopliae has the capability to differentiate into diverse cellular types (e.g. mycelia, conidia, appressoria and blastospores) during the infection cycle. In order to undergo such modifications the fungal cell wall structure suffers extensive remodeling which includes hydrolysis of its major component, chitin. This linear homopolymer of N-acetyl-β-D-glucosamine can be disrupted between its β-1,4-linkages by the enzymatic action of the glycoside hydrolase family 18 (GH18) proteins, known as chitinases (E.C. 3.2.1.14). The full enzymatic chitin hydrolysis to render N-acetylglucosamine is performed by chitinolytic systems, acting in a synergistic and consecutive way by the activities of chitinases and N-acetyl-glucosaminidases. A search in M. anisopliae strain E6 genome successfully identified 24 putative chitinases belonging to GH18 family which were further categorized into four subgroups (A, B, C and D) . Subgroup C chitinases (ChiMaC1, ChiMaC2, ChiMaC3 and ChiMaC4) are a distinct subgroup of GH18 proteins which presents the largest amino acid length among the family and also exhibit LysM domains. In order to better detail the roles of those genes among different cell types, the relative transcript levels from the 4 chitinase genes was evaluated by quantitative PCR (qPCR) over different conditions (mycelium growth on complete and minimal media, autolysed cultures, conidium, appressorium and blaspostore). chimaC1 and chimaC4 transcript levels were induced by growth on complete medium, chimaC2 and chimaC3 showed transcript level induction on autolytic conditions and chitin 1%, respectively. In general, sg C chitinases presented the lower transcript levels when comparing all chitinase transcripts. Single gene knock-outs from sg C chitinases are being developed by using Agrobacterium tumefaciens-mediated transformation of M. anisopliae with four vectors each containing the respective deletion cassette. Multiple phenotypic and genetic analyses will be performed in order to evaluate modifications induced by subgroup C chitinases gene knock-out.